Breast cancer is the most common malignancy, which occurs in women and the 2nd leading cause of cancer death. While the majority of breast cancers are sporadic (not inherited), approximately 10 percent are due to inherited mutations in the BRCA1 and BRCA2 genes. Population studies show that mutations in these genes lead to a lifetime risk of approximately 80 percent for breast cancer and 40-65 percent for ovarian cancer. Although physicians have previously relied on a high-risk family history of breast cancer to identify women at risk for carrying mutations in BRCA1/2, a recent study shows that 50 percent of carriers have no significant family history. Public health would be improved by identifying such carriers since there are an increasing number of therapeutic options, which can significantly reduce risk. However, conventional methods of detecting BRCA1/2 mutations based on direct sequencing are too expensive (approximately $3,000) and labor intensive for population screening. The objective of this project is to develop a cost-effective technology to screen for mutations in the BRCA1/2 genes. Two different methods will be developed and evaluated; both based on in vitro expression of peptides from overlapping segments of PCR amplified BRCA1/2 genomic DNA and mRNA. One approach utilizes a newly developed ELISA-based protein truncation test (ELISA-PTT) to detect chain-truncations, which constitute 90 percent of all mutations in BRCA1/2. In contrast to conventional protein truncation tests, ELISA-PTT eliminates the need for electrophoresis and radioactivity. A second approach, which again does not require electrophoresis or radiolabels, utilizes mass spectrometric analysis of in vitro expressed proteins (MASSIVE-PRO) to scan for all possible mutations, including amino acid substitutions. A key to this approach is the development of an in vitro expression system, which has very low levels of proteolytic activity. Preliminary studies by us demonstrate that both approaches are feasible and can offer a very low cost and high throughput alternative to full DNA sequencing. A key project scientist is Dr. Alex Garvin, an expert on BRCA1/2 protein truncation tests and developer of MASSIVE-PRO. The technology will be extensively evaluated in collaboration with Dr. Jessica K. Booker, Associate Director of the Molecular Diagnostics Laboratory at the University of North Carolina School Of Medicine (UNCSM) using a repository of validated genomic DNA and mRNA samples from breast cancer patients. During Phase II, an optimized system for screening BRCA1/2 mutations will be developed and clinically evaluated.